High glucose alters apoptosis and proliferation in HEK293 cells by inhibition of cloned BK_Ca channel

Abstract

It has been reported that diabetic vascular dysfunction is associated with impaired function of large conductance Ca(2+) -activated K(+) (BK(Ca) ) channels. However, it is unclear whether impaired BK(Ca) channel directly participates in regulating diabetic vascular remodeling by altering cell growth in response to hyperglycemia. In the present study, we investigated the specific role of BK(Ca) channel in controlling apoptosis and proliferation under high glucose concentration (25 mM). The cDNA encoding the α+β1 subunit of BK(Ca) channel, hSloα+β1, was transiently transfected into human embryonic kidney 293 (HEK293) cells. Cloned BK(Ca) currents were recorded by both whole-cell and cell-attached patch clamp techniques. Cell apoptosis was assessed with immunocytochemistry and analysis of fragmented DNA by agarose gel electrophoresis. Cell proliferation was investigated by flow cytometry assays, MTT test, and immunocytochemistry. In addition, the expression of anti-apoptotic protein Bcl-2, intracellular Ca(2+) , and mitochondrial membrane potential (Δψm) were also examined to investigate the possible mechanisms. Our results indicate that inhibition of cloned BK(Ca) channels might be responsible for hyperglycemia-altered apoptosis and proliferation in HEK-hSloα+β1 cells. However, activation of BK(Ca) channel by NS1619 or Tamoxifen significantly induced apoptosis and suppressed proliferation in HEK-hSloα+β1 cells under hyperglycemia condition. When rat cerebral smooth muscle cells were cultured in hyperglycemia, similar findings were observed. Moreover, the possible mechanisms underlying the activation of BK(Ca) channel were associated with decreased expression of Bcl-2, elevation of intracellular Ca(2+) , and a concomitant depolarization of Δψm in HEK-hSloα+β1 cells. In conclusion, cloned BK(Ca) channel directly regulated apoptosis and proliferation of HEK293 cell under hyperglycemia condition.

High-Resolution Live-Cell Imaging and Analysis by Laser Desorption/Ionization Droplet Delivery Mass Spectrometry

“…26–28 We induced apoptosis in HEK 293T cells by applying 35 mM ethanol for 20 min to the cell culture. Strong differences were apparent in both bright-field images and in the changes in the types and intensities of the peaks in the mass spectra (Figure 6).…”

BK channel β1-subunit deficiency exacerbates vascular fibrosis and remodelling but does not promote hypertension in high-fat fed obesity in mice

“…Therefore, BK channel deficiency-induced SMC proliferation could occur in the absence of pressure loading. [38,39] These may explain why high-fat-BK β1-knockout mice developed sever vascular remodeling and fibrosis even without increases in BP.…”

“…[41] It is not known if vascular BK β1-subunit deficiency affects extracellular matrix (collagen) degradation or synthesis, but cloned BK channels directly regulated apoptosis and proliferation of HEK293 cell under hyperglycemic conditions. [38] BK channels also mediate laminar shear stress-induced VSMC proliferation in cell culture. [39] Further studies will be needed to determine the mechanisms responsible for BK channel deficiency associated vascular remodeling and fibrosis in obesity.…”

“…This speculation is supported by the studies that high glucose inhibits BK channel α-subunit activity in BK α-subunit transfected HEK 293 cells. [38] Therefore, the impaired BK channel function in high-fat-wild-type mice could be caused by both downregulated BK β1-subunit expression and direct inhibition of BK channel α-subunit activity. Nevertheless, our studies show that BK β1-subunit dysfunction contributes to obesity associated systemic vascular dysfunction, remodeling and fibrosis.…”

Salidroside contributes to reducing blood pressure and alleviating cerebrovascular contractile activity in diabetic Goto-Kakizaki Rats by inhibition of L-type calcium channel in smooth muscle cells

“…As previously described [29], isolated VSMCs were cultured in DMEM (GIBCO, USA) supplemented with 20% FBS (HyClone, USA). To diminish the interferences of serum, the VSMCs were cultured in 5% FBS + 3% Insulin-Transferrin-Selenium (ITS) during the pharmacological experiments.…”